Skirball Institute of Biomolecular Medicine, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA. Electronic address: karim-jean.armache@med.nyu.edu.

Abstract

The maintenance of gene expression patterns during metazoan development is achieved, in part, by the actions of polycomb repressive complex 2 (PRC2). PRC2 catalyzes mono-, di-, and trimethylation of histone H3 at lysine 27 (H3K27), with H3K27me2/3 being strongly associated with silenced genes. We demonstrate that EZH1 and EZH2, the two mutually exclusive catalytic subunits of PRC2, are differentially activated by various mechanisms. Whereas both PRC2-EZH1 and PRC2-EZH2 are able to catalyze mono- and dimethylation, only PRC2-EZH2 is strongly activated by allosteric modulators and specific chromatin substrates to catalyze trimethylation of H3K27 in mouse embryonic stem cells (mESCs). However, we also show that a PRC2-associated protein, AEBP2, can stimulate the activity of both complexes through a mechanism independent of and additive to allosteric activation. These results have strong implications regarding the cellular requirements for and the accompanying adjustments in PRC2 activity, given the differential expression of EZH1 and EZH2 upon cellular differentiation.

(A) Western blot analysis of EZH2, EZH1, H3K27me1, H3K27me2, H3K27me3, Gapdh and total histone H3 levels from E14 mESCs WT cells or cells carrying EZH2-KO, B6 mESCs WT cells or cells carrying EZH1-KO or EZH1-KO/EZH2ΔSET, as indicated. (B-E) Histone methyltransferase assays. The details of HMT assay conditions as well as the generation of the nucleosome substrates are described in Materials & Methods. (n=3/data point). Data Plotted as Mean±SD.(B) Top, HMT assays containing PRC2-EZH1 or PRC2-EZH2 (15, 30, or 60 nM) using core nucleosomes, mononucleosomes, or chromatinized plasmids as substrates (300 nM), as illustrated on the panel. The levels of methylation on histone H3 are shown by autoradiography (Top image). Coomassie blue staining of SDS-PAGE gels containing nucleosomes (Middle image) or PRC2 components (Bottom image) was used to visualize the relative concentration of each component present in each reaction. Bottom, quantification of the relative amount of 3H-SAM incorporated into histone H3 after 60 min of incubation.(C) Top, HMT assays containing PRC2-EZH1 or PRC2-EZH2 (15 nM) using core nucleosomes (300 nM) as substrate with increasing amounts of 75 bp double-stranded (ds) DNA (200, 400, or 800 nM). Other panels are as described in (B).(D and E) Top, HMT assays containing PRC2-EZH1 or PRC2-EZH2 (15, 30, or 60 nM) using 300 nM of di-nucleosomes (D) or 12N oligo-nucleosomes (E) containing indicated lengths of linker DNA as substrates. Other panels are as described in (B).

Allosteric, SRM-dependent activation of PRC2-EZH2 is significantly more efficient than that of PRC2-EZH1

(A) Top, HMT assays containing PRC2-EZH1 or PRC2-EZH2 (15 nM) using core nucleosomes (Left) or chromatinized plasmids (Right) as substrates (300 nM) in the absence or presence of the H3K27me3 peptide, as indicated. Top/middle/bottom images are described in . Bottom, quantification of the relative amount of 3H-SAM incorporated into histone H3 after 60 min of incubation, (n=3/data point). Data Plotted as Mean±SD.(B) Top, Sequence alignment of the SRM domain of human EZH1 and human EZH2. Distinct residues between EZH1 and EZH2 (EZH1 C130 and EZH2 H129) are highlighted by blue box. Bottom, Structural depiction of the SRM domain of human PRC2-EZH2 with JARID2-K116me3 (111–121) peptide (modified from PDB:5HYN). His129 of EZH2, and Arg236 and Arg302 of EED are highlighted.(C) Left, HMT assays containing PRC2-EZH2, PRC2-EZH1, or PRC2-EZH2H129C (15 nM) using core nucleosomes (300 nM) as substrate in the absence or presence of the H3K27me3 peptide (50, 100, or 200 nM), as indicated. Other panels are as described in (A). Right, quantification of the relative amount of 3H-SAM incorporated into histone H3 after 60 min of incubation, (n=3/data point). Data Plotted as Mean±SD.(D) Western blot analysis of EZH2, EED, H3K27me1, H3K27me2, H3K27me3, Gapdh, and total histone H3 levels from E14 mESCs WT cells or cells carrying EZH2-KO rescued with EZH2WT or EZH2H129C mutant, as indicated. EV, empty vector.

(A) Top, HMT assays containing PRC2-EZH2 (60, 120, or 240 nM) in the absence or presence of H3K27me3 peptide, AEBP2WT, AEBP2KR(171–174/178–181)A, or both H3K27me3 and AEBP2WT (60, 120, or 240 nM) using H3K27me2 chromatinized plasmids (300 nM) as substrate. Top/middle/bottom images are described in . Note that the autoradiography signal (Top image) represents tri-methylation of H3K27. Bottom, quantification of the relative amount of 3H-SAM incorporated (H3K27me3) into histone H3 after 60 min of incubation, (n=2/data point). Data Plotted as Mean±SD.(B) HMT assays containing PRC2-EZH2P132S (60, 120, or 240 nM) in the absence or presence of AEBP2WT, AEBP2KR(171–174/178–181)A (60, 120, or 240 nM) using H3K27me2 chromatinized plasmids (300 nM) as substrate. Other panels are as described in (A).(C) HMT assays were performed as described in panel A except using PRC2-EZH1 (60, 120, or 240 nM).(D) HMT assays containing PRC2-EZH2 (Left) and PRC2-EZH1 (Right) performed in (here, Top image) were subjected to immunoblotting against H3K27me1, H3K27me2, and H3K27me3.